Aoki Kazuma, Ishii Yasuhiro, Matsumoto Ken, Tsujimoto Masafumi
Laboratory of Cellular Biochemistry, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan.
Nucleic Acids Res. 2002 Dec 1;30(23):5182-92. doi: 10.1093/nar/gkf638.
Cold-inducible RNA-binding protein (CIRP) was originally found in mammalian cells as a protein that is overexpressed upon a temperature downshift. Recently, we identified a Xenopus homolog of CIRP, termed xCIRP2, as a major cytoplasmic RNA-binding protein in oocytes. In this study we found by yeast two-hybrid screening that the Xenopus homolog of protein arginine N-methyltransferase 1 (xPRMT1) interacted with xCIRP2. We found that an arginine- and glycine-rich region of xCIRP2, termed the RG4 domain, was a target of xPRMT1 for methylation in vitro. xCIRP2 expressed in cultured cells accumulated in the nucleus as does mammalian CIRP. Interestingly, the RG4 domain was necessary for nuclear localization of xCIRP2. RG4-mediated nuclear accumulation of xCIRP2 was diminished in the presence of transcription inhibitors, suggesting that nuclear localization of xCIRP2 was dependent on ongoing transcription with RNA polymerase II. Analysis of interspecies heterokaryons revealed that xCIRP2 was capable of nucleocytoplasmic shuttling and the RG4 domain functioned as a nucleocytoplasmic shuttling signal. Methylation by overexpressed xPRMT1 caused cytoplasmic accumulation of xCIRP2. Possible implications of the relationship between regulation of intracellular localization and multiple functions of xCIRP2 will be discussed.
冷诱导RNA结合蛋白（CIRP）最初是在哺乳动物细胞中发现的一种在温度下降时过度表达的蛋白质。最近，我们鉴定出一种非洲爪蟾CIRP的同源物，称为xCIRP2，它是卵母细胞中的一种主要细胞质RNA结合蛋白。在本研究中，我们通过酵母双杂交筛选发现，蛋白质精氨酸N-甲基转移酶1（xPRMT1）的非洲爪蟾同源物与xCIRP2相互作用。我们发现，xCIRP2中一个富含精氨酸和甘氨酸的区域，称为RG4结构域，是xPRMT1在体外进行甲基化的靶点。在培养细胞中表达的xCIRP2像哺乳动物CIRP一样在细胞核中积累。有趣的是，RG4结构域是xCIRP2核定位所必需的。在存在转录抑制剂的情况下，RG4介导的xCIRP2核积累减少，这表明xCIRP2的核定位依赖于RNA聚合酶II的持续转录。种间异核体分析表明，xCIRP2能够进行核质穿梭，并且RG4结构域起到核质穿梭信号的作用。过表达的xPRMT1进行的甲基化导致xCIRP2在细胞质中积累。我们将讨论细胞内定位调控与xCIRP2多种功能之间关系的可能意义。
